Low incidence of coronary angiography in the evaluation process of the potential heart donor.

INTRODUCTION: We investigated the practice of coronary angiography (CA) on donor hearts. PATIENTS AND METHODS: Between January 1, 2000, and December 31, 2010, all reported organ donors aged <66 years were analyzed retrospectively. Donor charts were evaluated regarding a performed CA, its outcome, the timing of CA during the evaluation process, and reasons for organ refusal. The percentage of positive CA studies in organ donors aged ≥45 years was also evaluated. RESULTS: Of 292 reported organ donors, 152 organ donor hearts were declined (group 1), and 140 hearts (group 2) were transplanted. Of the 152 declined hearts, 91 hearts were found not suitable for organ offer, and 61 were not successfully allocated or were refused by Eurotransplant. CA was conducted in 17 organ donors (5.8%). In 6 donors, a previous CA was reported (all had pathologic findings), and in 11 donors, a donor CA was performed, indicating 4 pathologic and 7 negative findings (54.5% of the hearts evaluated by donor CA were transplanted). No complication or delay of the donation process was reportedly related to donor CA. CONCLUSIONS: Special emphasis and implementation of recommendations for CA to be part of the evaluation of donor organs seem necessary.

Serum albumin, subjective global assessment, body mass index and the bioimpedance analysis in the assessment of malnutrition in patients up to 15 years after liver transplantation.

BACKGROUND: The subjective global assessment (SGA) or the body mass index (BMI) is used to determine the nutritional state after LTX. Bioelectrical impedance analysis (BIA) is used as tool to determine body composition by nutritional care professionals. METHODS: BIA, SGA, BMI, and serum albumin (SA) levels were performed to assess malnutrition following liver transplantation. BIA measurement was used as reference standard to determine existing malnutrition. A phase angle (PA) <5 was used to define potentially existing chronic disease-related malnutrition as a standard. All other measured parameters were compared with respect to their prognostic accuracy regarding the prediction of malnutrition as compared to the mentioned standard. RESULTS: Seventy-one recipients (51 men, 20 women) were included. Median age was 58, weight 77 kg, BMI 26 kg/m(2) , PA 4.1°, and SA 4.3 g/dL. According to the Nutritional Risk Screening 2002, 9.4% (6/71), to BMI 15.4% (11/71), to SA 30.9% (22/71), and to BIA 36.5% (28/71) of the patients were malnourished. PA did not correlate with BMI or NA, there was a significant correlation with SA (p = 0.001). Univariate analysis revealed SA as independent predictor for malnutrition. ROC analysis for all parameters revealed a significantly (p < 0.05) better area under the receiver operating characteristic curve for SA (0.812) than for BMI (0.603) for the prediction of malnutrition. CONCLUSION: SGA or BMI calculation alone does not suffice to evaluate the nutritional status. SA seems to play a crucial role in the prediction of severe disease-related malnutrition in this special patient cohort.

Isoprostanes as markers of oxidative stress-induced cell damage in porcine islet cell isolation.

INTRODUCTION: Mechanical stress and reagents used during the isolation and purification process as well as digestion time and temperature can alter the success of porcine islet cell (PIC) isolation. This study aimed to characterize the occurrence of isoprostanes during PIC isolation using a modified automated Ricordi method and to evaluate their influence on PIC isolation outcome. METHODS: Porcine pancreatic tissue was harvested at the local slaughter house, and 10 PIC isolations were performed using a modified automated Ricordi method. As positive controls for tissue damage-associated oxidative stress, six consecutive PIC isolations were performed in the presence of 1 mug lipopolysaccharide (LPS). PIC were purified by density gradient centrifugation using the Lymphoprep density gradient. Isoprostane measurement was performed using enzyme-linked immunosorbent assay. RESULTS: The final yield of viable and pure PICs in the experimental group was 3479 +/- 542 IEQ/g pancreas, and the LPS group yielded lower cell numbers compared to the experimental group. Isoprostane levels were significantly elevated in the LPS group as compared to the experimental group at all time points during the isolation from the beginning of the digestion process. DISCUSSION: PIC isolation and purification results significantly differed in the two experimental groups, underlining the negative effects of oxidative stress on PIC viability and purity, which impact negatively on PIC transplantation success.

Preoxygenation of different preservation solutions for porcine pancreas preservation.

INTRODUCTION: Organ preservation quality impacts porcine islet cell isolation and transplantation success. Among several preservation methods, the two-layer method is promising, but technically demanding and fails to deliver sufficient oxygen. The use of hyperbaric oxygenation may be an easier, more effective method to supply high partial pressure of oxygen (pO(2)) for organ storage. Therefore, the aim of this study was to test the capability of preoxygenation of various preservation solutions with HBO to maintain high pO(2) levels. METHODS: University of Wisconsin (UW), Custodiol, Perfadex, or Celsior solutions were preoxygenated in a pressure chamber. NaCl served as the control. pO(2) levels were measured at defined times. The oxygen storage capability was evaluated by leaving the storage bottles open for 2 minutes. RESULTS: It was feasible to preoxygenate preservation solutions. The best solution to maintain high pO(2) tensions was Perfadex, followed by Celsior, and UW. DISCUSSION: The greater the amount of oxygen in the preservation solution, the more oxygen can be delivered to the preserved pancreas. Further studies on the influence of preoxygenated preservation solutions on the porcine pancreas are warranted to improve organ quality, porcine islet cell isolation, and transplantation success.

Ductal injection of University of Wisconsin solution prior to pancreas preservation prevents oxidative cell damage.

INTRODUCTION: Several studies have been carried out investigating different preservation methods and preservation solutions for the pancreata of various species. Attention has to be drawn to the extreme vulnerability of porcine pancreata (PP) to oxidative stress due to the lack of endogenous antioxidants. This study sought to evaluate the influence of cannulation and infusion of different volumes of University of Wisconsin (UW) solution immediately after organ retrieval on PP organ quality. METHODS: PP from 24 slaughterhouse pigs were harvested with immediate cannulation of the pancreatic duct for infusion of 10 mL, 20 mL, 50 mL, or 100 mL UW solution. The organs were stored in cold UW solution. Control organs were only stored in UW. After 6 hours of cold ischemia, tissue and supernate samples were analyzed for markers of oxidative cell damage, adenosine triphosphate (ATP) levels, and occurrence of apoptosis. RESULTS: The fewest apoptotic cells were detected in the PP infused with 50 mL UW via the pancreatic duct (PP 50) as compared with all other groups. Oxidative cell damage was lowest and ATP levels were highest in the PP 50 group. DISCUSSION: Because PP 50 showed significantly better results when compared with all other groups, we suggest that infusion of 50 mL UW via the pancreatic duct immediately after organ retrieval may be useful to minimize oxidative cell damage and cell death in PP.

The disease relevance of human hepatocellular xenograft models: molecular characterization and review of the literature.

In recent years a number of new therapeutics has been developed that were not general toxins and inhibitors of cell division like classical chemotherapeutics, but were designed to target a specific pathway. A prerequisite for this development was the comprehensive characterization of molecular alterations occurring in human hepatocellular carcinoma (HCC). However, while much knowledge of the molecular pathogenesis of human HCC has been gained, the model systems used to test the functional relevance of these alterations and applied for preclinical evaluation of drug candidates are still poorly characterized. In this paper, we reviewed the literature about several commonly used HCC cell lines and xenotransplantation models and present our own data on the molecular characterization of these. Results obtained demonstrate that it is important to have a sound knowledge of the specific molecular constitution of the experimental model and to carefully evaluate the functional status of the pathway of interest. For this reason, we make the gene expression profiles publicly available to help researchers making an informed decision about which model to use.

Sensitive serum parameters for hepatocellular carcinoma recurrence monitoring after liver transplantation: a case report.

Liver cell malignancy can be seen as one of the most common indications for hepatic transplantation, but the recurrence potential of the disease significantly limits its beneficial effects. Hepatic factors influencing the recurrence rate, such as nodule size and criteria wherein transplantations are expedited, are still investigated. Pretransplant intraarterial or percutaneous treatment seem to be predictive for recurrence-free patient survival. Early detection of malignancies via serum parameters as a prognostic factor seems promising. This article reports a special case, where despite an elevated, sensitive, serum marker profile, no HCC recurrence was detected over a 2-year follow-up.

Cryopreservation of freshly isolated porcine islet cells.

INTRODUCTION: The use of xenogenic islet cells may be a possibility to overcome the shortage of human donor organs to treat diabetes. Microencapsulation seems to be a promising method for immunoprotection. Since isolation, purification, encapsulation, and transplantation of islet cells are labor intensive, cryopreservation has emerged as an attractive system of islet banking. The aim of this study was to determine the influence of three different freezing media (FM) on viability of freshly isolated porcine islet cells (FIPIC). METHODS: FIPIC were isolated using a modified Ricordi method and purification performed using a Lymphoprep density gradient. Viability of FIPIC prior to freezing and after thawing was determined using the MTT-based Cell Growth Determination Kit. Insulin production was detected using enzyme-linked immunosorbent assay. Three different FM containing dimethylsulfoxide (DMSO) or glycerol and sucrose were used for cryoprotection of FIPIC. RESULTS: Isolation and purification of FIPIC resulted in 95% +/- 1.3% viability and 97% +/- 1.4% purity. Cryopreservation with FM I (containing DMEM, FCS, DMSO) yielded 98.4% and FM III (containing DMEM, FCS, glycerol) 93.1% viability, whereas only 85.6% were alive when cryoprotection is performed with FM II (containing DMSO, BM). Glucose stimulation revealed a loss of 2.8% and 1.9% of insulin secretion per microgram DNA when working with FM I and FM III, but a decrease in glucose-dependent insulin secretion of 7.8% (P < .05) when FIPIC were stored in FM II. DISCUSSION: Low concentrations of DMSO or the use of glycerol and sucrose seem to be equivalent to cryopreserve FIPIC.

Cryopreservation of insulin-producing cells microencapsulated in sodium cellulose sulfate.

INTRODUCTION: Diabetes mellitus may be treated with pancreatic islet cell transplantation. The use of xenogenic islet cells may overcome the shortage of human donor organs. Microencapsulation seems to be a promising method for immunoprotection. Since isolation, purification, encapsulation, and transplantation of islet cells are labor-intensive, cryopreservation has emerged as an attractive system for islet banking. In this study sodium cellulose sulfate (NaCS), a novel method for microencapsulation of islet cells, was tested for its capability to protect cells during cryopreservation. METHODS: HIT-T15 cells were microencapsulated in NaCS. Cells were frozen and thawed using three different media containing varying amounts of dimethylsulfoxide (DMSO) and glycerol. Cell viability and cell growth were monitored using 3-(-4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide before freezing and 1 week after thawing. RESULTS: NaCS did not show any negative impact on the growth rates of encapsulated HIT-T15 cells compared with nonencapsulated controls. Nonencapsulated cells were adequately cryopreserved by both DMSO- and glycerol-containing freezing media. DMSO was not suitable for cryopreservation of encapsulated HIT-T15 cells, whereas glycerol seemed to produce no considerable cell loss during freezing and thawing. DISCUSSION: Islet banking of cells encapsulated in NaCS was feasible. Microencapsulation did not harm islet cell recovery. As NaCS is less immunogenic and more biocompatible than other materials used for microencapsulation, it may be a promising method for immunoisolation of islet cells to replace the endocrine pancreas in a physiological way.

Morphological and functional characterization of a pancreatic beta-cell line microencapsulated in sodium cellulose sulfate/poly(diallyldimethylammonium chloride).

BACKGROUND: Late diabetic complications cannot be prevented totally by current antidiabetic strategies. Therefore, new therapeutic concepts of insulin replacement such as pancreas transplantation are evolving. Due to the shortage of human donor organs, transplantation of microencapsulated xenogeneic pancreatic islet cells has attracted considerable attention. Sodium cellulose sulfate/poly(diallyldimethylammonium chloride) (NaCS/PDADMAC) is a material with favorable biogenic properties that has been used for microencapsulation of various cell types. However, there are no data on the suitability of NaCS/PDADMAC for microencapsulation of pancreatic beta-cells. MATERIAL AND METHODS: Cell growth and viability of NaCS/PDADMAC-microencapsulated HIT-T15 cells, an immortalized hamster pancreatic beta-cell line, were assessed using a dimethylthiazol-diphenyltetrazoliumbromide (MTT)-based cell growth determination kit and apoptosis was detected by antibodies against activated caspase 3. Glucose-dependent insulin secretion was assessed with ELISA and the uptake of glucose was measured using fluorescence-labeled glucose. RESULTS: Statistical analysis revealed no differences in glucose-dependent cell proliferation, insulin secretion and glucose uptake between non-microencapsulated and microencapsulated HIT-T15 cells. Stimulation of HIT-T15 cells with glucose (100 mg/ml) resulted in a biphasic insulin secretion response. CONCLUSION: Microencapsulation of HIT-T15 cells in NaCS/PDADMAC does not influence cell proliferation, insulin secretion and glucose uptake. Our results indicate that NaCS/PDADMAC is well suited for microencapsulation of pancreatic beta-cells.

Porcine islet cells microencapsulated in sodium cellulose sulfate.

One hundred fifty million people suffer from diabetes mellitus worldwide. Modern exogenous insulin therapy cannot prevent late complications. Islet cell transplantation could be a sufficient therapeutic option but the shortage of human organs limits this option. The use of xenogeneic porcine islet cells may also be a viable alternative. One way to manage hyperacute rejection is by the protection of xenogeneic cells from the immune system by microencapsulation. In this study sodium cellulose sulfate (NaCS) was evaluated as a material for encapsulation. An insulin-producing cell line (HIT-T15) was established in our laboratory. Glucose-dependent insulin production and cell growth were monitored. Cells were encapsulated with NaCS by Austrianova, Vienna. The insulin production and mitosis rate were examined. Cell growth and insulin production by HIT-T15 cells affected the glucose levels in the nutrient solution. Cell viability and glucose-dependent insulin production were not influenced by NaCS. Encapsulation with NaCS is feasible and it could be shown that the material is permeable to nutrients and metabolic side products. The encapsulated cells are able to detect the glucose concentration in the nutrient solution and to react in a proper way by producing insulin. Encapsulation with NaCS, which is more biocompatible and less immunogenic than other materials, seems to be a promising method for immunoisolation of porcine beta cells for xenotransplantation to replace the endocrine pancreas in a physiologic way.

Nonnephrotoxic immunosuppression in patients after liver transplantation.

INTRODUCTION: Calcineurin inhibitor (CI)-associated renal impairment and renal failure after liver transplantation has been recognized since the early days of its use. Various strategies have been used to prevent or slow down the progression of renal dysfunction in liver transplant recipients, but did not succeed. In this report, we describe the course of renal function of 58 stable liver transplant recipients and compared 2 groups with different immunosuppressive protocols. METHODS: In the study group, 22 patients at various intervals from liver transplantation were included. The immunosuppressive therapy consisted of Sirolimus (SRL). Additional all patients except 2 received Mycophenolate Mofetil (MMF) and 14 of them also received Tacrolimus. Patients of the control group (36 patients) had an immunosuppressive therapy with calcineurin inhibitors. Patients were monitored for creatinine monthly and creatinine clearance (CCr) every sixth month. Risk factors for renal dysfunction were evaluated. RESULTS: After introduction of SRL in patients with renal impairment and after a mean follow-up time of 12 (2-26) months, there was a decrease of 28.3% in mean creatinine and of 41.8% in mean urea. We observed an improvement of renal function in all patients initially after introduction of SRL. In the control group, in comparison to preoperative levels, there was an increase of 27.5% in mean creatinine and of 13.3% in mean urea after a mean follow-up time of 3.6 years with CI therapy. CONCLUSION: The results of our retrospective study showed that with SRL renal impairment could be stopped and renal function could be improved. We suggest administering immunosuppressive therapy with SRL in combination with low dose Tacrolimus and/or MMF for patients with renal impairment.

Center experience in liver transplantation (LTX): management of dermal side effects caused by sirolimus.

INTRODUCTION: Sirolimus improves post transplant maintenance therapy in LTX. Dermal side effects causing pain and discomfort can limit patients' compliance. The package insert mentions such skin disorders as acne and rash. One case of sirolimus-induced leucocytoclastic vasculitis is reported in the literature. METHODS: From July 1998 to October 2003, Sirolimus was implemented in the immunosuppressive protocol in 23 out of 60 liver recipients. Sirolimus target levels are between 3 and <10 ng/dl. Combination with a calcineurinblocker and/or MMF (mycophenolate mofetil) depending on liver function and creatinine is standard. Weekly patient monitoring in the first month after discharge included physical examination, blood samples and immunosuppresant trough levels. Biopsies were taken from untypical efflorescences. RESULTS: Three patients with non-specific effloresces were reported: one with leucocytoclastic vasculitis and one with exfoliate forearm dermatitis required change of medication while one perivascular lymphocytic eosinophilic dermatitis subsided after dose reduction. In three cases of mouth ulcer, trough levels exceeded 10 ng/dl and in six patients acne diminished after dose reduction. Eighteen out of 23 patients are still receiving sirolimus. Reasons for removal from the study were incompliance and incompatibility. Two patients died. DISCUSSION: Immunosuppressants inevitably produce side effects in TX recipients. The positive management of troublesome side effects contributes importantly to compliance and patient survival.

[Consensus-recommendations for sirolimus in liver transplantation]. / Konsensusempfehlung zum Einsatz von Sirolimus in der Lebertransplantation.

Sirolimus is an m-TOR inhibitor without renal side effects and potentially protects against the development of malignancy. Due to a higher incidence of complications in two trials and an official warning in the drug information, the use of Sirolimus in liver transplantation is limited. The participants of this consensus meeting had to analyse and evaluate the literature with respect to the potential role of Sirolimus in liver transplantation. This consensus statement follows the scheme normally employed for the presentation of guidelines including the grading of evidence (1a-5) and the extent of recommendation (A-C). Moreover, the consensus included the experience of the authors with respect to the handling of Sirolimus after liver transplantation.

Dyslipidemia during sirolimus therapy in patients after liver transplantation.

INTRODUCTION: Sirolimus (SRL) is an immunosuppressive agent of potential benefit in clinical liver transplantation (LTX). One of the major side effects of SRL is hyperlipidemia, which is reported in up to 44% of patients. In this report, we describe the lipid profiles of 20 stable liver transplant recipients who received SRL for immunosuppression. METHODS: The study group received SRL in combination with tacrolimus and/or mycophenolate mofetil (MMF). The control group was administered calcineurin inhibitor (CI) and MMF. Fasting serum cholesterol level, high-density lipoproteins (HDL) and low-density lipoproteins (LDL) were measured regularly. Furthermore, the total cholesterol/HDL ratio and the LDL/HDL ratio were evaluated. Diabetes and hypertension were monitored as well. RESULTS: In the SRL group, hypercholesterolemia was found in three patients (15%) and hypertriglyceridemia in two patients (10%). There was no marked difference from the control group, although a higher association of SRL with hyperlipidemia was described in the literature. Furthermore, HDL and LDL levels were similar in both groups, as well as total cholesterol/HDL ratio and LDL/HDL ratio. Diabetes and hypertension had a similar incidence in both the groups. Thus, there was no difference concerning the cardiovascular atherosclerosis risk between the immunosuppressive protocol with SRL or with CI. DISCUSSION: The results of our retrospective study demonstrated that the immunosuppressive regimen can potentially influence the incidence of hyperlipidemia in patients after LTX. SRL in combination with tacrolimus and/or MMF had no higher incidence of hyperlipidemia than CI and MMF. The combination of immunosuppressive therapy with low dose and low levels of each immunosuppressive agent could decrease the risk of atherosclerosis and its complications in long-term survivors after LTX.

Experiences in liver transplantation for hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is one of the most common cancers worldwide. Beside surgical resection, orthotopic liver transplantation (OLT) is not only effective but also the only potentially curable treatment in selected cases of small tumors. We report our experience in 11 male patients transplanted for HCC from August 1998 to July 2002. Selection criteria for OLT were unresectability of the hepatic tumor and severity of the underlying liver disease. The tumor diagnosis was confirmed by histology, imaging techniques, and tumor markers. All patients received an orthotopic liver allograft using a modified piggyback technique. Six of the 11 patients are alive; one died due to acute rejection and four died from recurrent disease. In all four patients with recurrent disease, vascular invasion was shown histologically, whereas only one patient without evidence of recurrence showed vascular invasion. To prevent recurrence after OLT the immunosuppressive regime was adjusted to the underlying disease by early cessation of prednisolone and reduction in the long-term exposure to immunosuppressive drugs. Patients were screened for recurrence by ultrasound and computed tomography. Recurrent HCC were treated symptomatically. OLT is an effective treatment for subgroups of patients with HCC. It might be possible to downstage the liver tumor by chemoembolization and/or radiofrequency ablation and allow the patients to wait for a suitable donor. After OLT the early withdrawal of prednisolone and the reduction of other immunosuppression is feasible. In conclusion, OLT can be a potentially curative therapy for HCC.

Occurance of apoptosis during ischemia in porcine pancreas islet cells.

PURPOSE: Pancreas islet transplantation is a potential treatment of diabetes mellitus and porcine organs provide an easily available source of cells. Unfortunately quality and quantity of isolated islets are still not satisfactory. Apoptosis occurs in freshly isolated islets and plays a significant role in early graft loss. We evaluated the influence of four storage solutions on porcine pancreas islets. METHOD: After warm ischemia of 15-20 minutes 12 organs were stored in 4 cold preservation solutions: Histidine-Tryptophan-Ketoglutarate solution (HTK), Hank's buffered saline solution (HBSS), University of Wisconsin (UW) solution and Ringer-Lactate (R). After cold ischemia for 100 minutes, organs were fixed in 3% formalin. Apoptotic cells were counted on hematocylin-eosin stainings. RESULTS: Most apoptotic cells were found in organs stored in R. Low numbers were found in the other groups. The difference between organs stored in R and organs stored in UW, HTK, or HBSS was highly significant. No significant difference could be found between UW, HTK and HBSS. CONCLUSION: Cold and warm ischemia of the pancreas seems to induce apoptosis in islet cells. Preservation solutions cause less apoptosis than electrolyte solution. No significant differences could be found among the preservation solutions.